Use of elsiglutide to treat gastrointestinal mucositis including chemotherapy-induced diarrhea

ABSTRACT

The invention relates to the use of elsiglutide to prevent or reduce the occurrence of gastrointestinal damage caused by chemotherapeutic agents, including gastrointestinal mucositis and chemotherapy-induced diarrhea (CID).

FIELD OF THE INVENTION

The invention relates to the use of elsiglutide to protect against andprevent gastrointestinal organ-specific toxicities induced bychemotherapeutic agents. In particular, this invention relates to theuse of elsiglutide to prevent and reduce the occurrence or severity ofgastrointestinal mucositis, and its clinical manifestations includingchemotherapy-induced diarrhea.

BACKGROUND OF THE INVENTION

Gastrointestinal (GI) damage and dysfunction are well-known side effectsof cancer-chemotherapy treatments and can be debilitating andpotentially life threatening (Richardson and Dobish, J Oncol PharmPractice 2007; 13:181-198; Grem et al., J Clin Oncol 1987; 5(10):1704;Petrelli et al., J Clin Oncol 1989; 7(10):1419-1426). Chemotherapyadministration is frequently associated with mucositis, diarrhea(chemotherapy-induced diarrhea (CID), bacterial translocation,malabsorption, abdominal cramping, gastrointestinal bleeding andvomiting. These side effects are clinical consequences of the structuraland functional damage of the intestinal epithelium and frequently makeit necessary to decrease the dose and frequency of chemotherapy, thusnegatively affecting the overall clinical outcome of the patients. Theintestinal mucositis and diarrhea can result in severe dehydration,electrolyte imbalances, sepsis due to bacterial translocation,cardiovascular instability, and renal insufficiency. Each of thesefactors contribute to a high morbidity and mortality associated with CID(Rubenstein et al., Cancer 2004; 100(9 Suppl):2026-2046; Rapoport etal., J Clin Oncol 1999; 17(8):2446-2453; Petrelli et al., J Clin Oncol1987; 5(10):1559-1565).

The stem cells of the small intestinal mucosa are particularlysusceptible to the cytotoxic effects of chemotherapy due to their rapidrate of proliferation (Keefe et al., Gut 2000; 47: 632-7).Chemotherapy-induced damage to the small intestinal mucosa is referredto as gastrointestinal mucositis and is characterized by absorptive andbarrier impairments of the small intestine. For example, it has beenshown that the broadly used chemotherapeutic agents 5-fluorouracil(5-FU), irinotecan and methotrexate increase apoptosis leading to villusatrophy and crypt hypoplasia in the small intestine of rodents (Keefe etal., Gut 47: 632-7, 2000; Gibson et al., J Gastroenterol Hepatol.September; 18(9):1095-1100, 2003; Tamaki et al., J Int Med Res.31(1):6-16, 2003). Chemotherapeutic agents have been shown to increaseapoptosis in intestinal crypts at 24 hours after administration andsubsequently to decrease villus area, crypt length, mitotic count percrypt, and enterocyte height three days after chemotherapy in humans(Keefe et al., Gut 2000; 47: 632-7). Thus, structural changes within thesmall intestine can lead directly to intestinal dysfunction and in somecases diarrhea.

Gastrointestinal mucositis after cancer chemotherapy is an increasingproblem that is essentially untreatable once established, although itgradually remits. Studies conducted with the commonly used cytostaticcancer drugs 5-FU and irinotecan have demonstrated that efficaciousdoses of these drugs predominantly affects structural integrity andfunction of the small intestine while the colon is less sensitive andmainly responds with increased mucus formation (Gibson et al., JGastroenterol Hepatol. September; 18(9):1095-1100, 2003; Tamaki et al.,J Int Med Res. 31(1):6-16, 2003).

Reported data about the frequency of CID and its severity can varysignificantly and have been derived mostly from clinical trials oncolorectal cancer treatments. Data from the late 1990s demonstrated CIDincidence to vary from a general 10-20% to the 50-80% found incolorectal cancer patients treated with 5-fluorouracil (5-FU) oririnotecan (with up to 30% of these patients experiencing severediarrhea, a life-threatening condition that may lead to hospitalization,and death in 2-5% of cases) (see Stein et al., Ther Adv Med Oncol 2010,2: 51-63; Gibson and Stringer, Curr Opin Support Palliat Care 2009, 3:31-35; Benson et al., J Clin Oncol 2004, 22: 2918-26; Conti et al., JClin Oncol 1996, 14: 709-15; Leichman et al., J Clin Oncol 1995, 13:1303-1311; Rothenberg et al., Cancer 1999, 85: 786-95). Tworetrospective studies have also been conducted that confirmed highestfrequencies of diarrhea from chemotherapy regimens based on 5-FU,irinotecan, capecitabine and/or oxaliplatin (Arbuckle et al., TheOncologist 2000, 5: 250-9; Goldberg et al., J Support Oncol 2005, 3:227-32). Importantly, some authors argue that CID events may beunderreported as well as under-recognized and underestimated in theclinical setting (Arbuckle et al., The Oncologist 2000, 5: 250-9;Cirillo et al., Annals of Oncology 2009, 20: 1929-35). This seems to beconfirmed by the large variability of frequencies reported inliterature.

Generally, CID is a dose-related adverse effect engendered by amulti-factorial process, through which the acute cytotoxic damage to theintestinal mucosa (including loss of intestinal epithelium, superficialnecrosis and inflammation of the bowel wall) causes an imbalance betweenabsorption and secretion in the small intestine (Stein et al., Ther AdvMed Oncol 2010, 2: 51-63; Gibson and Stringer, Curr Opin Support PalliatCare 2009, 3: 31-35; Keefe, Curr Opin Oncol 2007, 19: 323-27). Exceptfor irinotecan-induced early onset diarrhea, a dose-dependent adversereaction occurring within 24 hours after the drug administration, theonset of CID episodes generally occurs a few days followingadministration, at all dose levels. For irinotecan, the median time tolate-CID onset is reported between 6 to 11 days following administration(Stein et al., Ther Adv Med Oncol 2010, 2: 51-63). Besides standardcytotoxic agents, recent findings also highlight that biologicaltargeted chemotherapeutic agents (such as tyrosine-kinase inhibitors)frequently cause diarrhea, which may occur in up to 60% of patientstreated with certain drugs, such as lapatinib (Stein et al., Ther AdvMed Oncol 2010, 2: 51-63; Lowell, 2012 MASCC/ISOO InternationalSymposium on Supportive Cancer Care). Therefore, in the future, morepatients with non-gastrointestinal cancers might also experiencetreatment-induced diarrhea.

The prompt assessment, diagnosis and implementation of appropriatemanagement strategies are essential for the prevention of potentiallysevere and also fatal consequences of CID (Benson et al., J Clin Oncol2004, 22: 2918-26; Maroun et al., Curr Oncol 2007, 14: 13-20; CommonTerminology Criteria for Adverse Events (CTCAE) Version 4.0 Published:May 28, 2009 (v4.03: Jun. 14, 2010):http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_(—)4.03_(—)2010-06-14_QuickReference_(—)8.5×11.pdf).In literature, the clinical assessment of diarrhea severity is mostcommonly measured by the NCI-CTC criteria, which define 5 severitydegrees of diarrhea, based on the number of bowel movements experiencedper day above baseline (Common Terminology Criteria for Adverse Events(CTCAE) Version 4.0 Published: May 28, 2009 (v4.03: Jun. 14, 2010):http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_(—)4.03_(—)2010-06-14_QuickReference_(—)8.5×11.pdf).Although the NCI-CTC criteria do not evaluate stool volume andconsistency, or diarrhea duration, this scale is widely accepted andused in clinical practice and clinical research (Saltz L B,Understanding and managing chemotherapy-induced diarrhea. J SupportOncol 2003, 1:35-46). Furthermore, several studies have accentuated thepsychosocial consequences of CID and suggested the importance ofevaluating the patient's perception and quality of life in order toachieve a comprehensive diagnostic assessment (Chen et al., Cancer 2010,116: 1879-86; Flores et al., Gastrointest Cancer Res 2012, 5:119-24).

Current pharmacologic treatments for manifestations of gastrointestinalmucositis such as CID are palliative at best; they help to control andtreat symptoms rather than to prevent the onset of diarrhea.Non-analgesic opiates such as loperamide are commonly employed becauseof their ability to promote intestinal fluid re-absorption and to slowdown intestinal motility. Loperamide is commonly used as a standardfirst-line therapy for Grades 1-2 diarrhea. Octreotide, a syntheticsomatostatin analogue has been shown to reduce the secretion of someintestinal hormones and promote intestinal absorption of fluids andelectrolytes. While it is not approved for this use, and has inadequateevidence demonstrating its utility, octreotide is often used for secondline treatment of loperamide refractory CID or for grades 3-4 diarrhea.Parenteral hydration and electrolyte supplementation and in severecases, total parenteral nutrition are used as well. See, e.g., Benson etal., J Clin Oncol 2004; 22(14):2918-2926. Other therapeutic options areavailable and under study, such as probiotics, but are not currentlyrecommended for practice (Benson et al., J Clin Oncol 2004;22(14):2918-2926; Muehlbauer et al., Clin J Oncol Nurs 2009, 13:336-41).

Because the currently available therapies for CID only offer symptomaticrelief, there is a great medical need for agents addressing the rootcauses of the disease that prevent or decrease the intestinal mucosaldamage caused by chemotherapy.

Glucagon-like-peptide-2 (GLP-2) is a 33-amino-acid peptide released fromthe post-translational processing of proglucagon in the enteroendocrineL cells of the intestine and in specific regions of the brainstem. It isco-secreted together with glucagon-like peptide 1 (GLP-1), oxyntomodulinand glicentin, in response to nutrient ingestion. GLP-2 inducessignificant growth of the small intestinal mucosal epithelium via thestimulation of stem cell proliferation in the crypts and inhibition ofapoptosis on the villi (Drucker et al. Proc Natl Acad Sci USA. 1996,93:7911-6). GLP-2 also inhibits gastric emptying and gastric acidsecretion (Wojdemann et al. J Clin Endocrinol Metab. 1999, 84:2513-7),enhances intestinal barrier function (Benjamin et al. Gut. 2000,47:112-9.), stimulates intestinal hexose transport via the upregulationof glucose transporters (Cheeseman, Am J Physiol. 1997, R1965-71), andincreases intestinal blood flow (Guan et al. Gastroenterology. 2003,125, 136-47).

The demonstrated specific and beneficial effects of GLP-2 in the smallintestine has raised much interest as to the use of GLP-2 in thetreatment of intestinal disease or injury (Sinclair and Drucker,Physiology 2005: 357-65). Furthermore GLP-2 has been shown to prevent orreduce mucosal epithelial damage in a wide number of preclinical modelsof gut injury, including chemotherapy-induced mucositis,ischemia-reperfusion injury, dextran sulfate-induced colitis and geneticmodels of inflammatory bowel disease (Sinclair and Drucker, Physiology2005:357-65).

GLP-2 is secreted as a 33 amino acid peptide having the sequenceHADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 2). It is rapidly cleavedat the Alanine (A) in position 2 of the N-terminus to the inactive humanGLP-2 (3-33) by the enzyme dipeptidyl peptidase-4 (DPP IV). This rapidenzymatic degradation of GLP-2(1-33), in addition to renal clearanceresults in a half-life of about 7 minutes for the peptide (Tavares etal., Am. J. Physiol. Endocrinol. Metab. 278:E134-E139, 2000).

U.S. Pat. Nos. 7,745,403 and 7,563,770 disclose GLP-2 analogues whichcomprise one of more substitutions as compared to wild-type GLP-2. Oneof the described GLP-2 analogues is ZP1846 (elsiglutide). A comparisonof the sequences of GLP-2 and elsiglutide is provided below:

elsiglutide: (SEQ ID NO: 1) HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK GLP-2: (SEQ ID NO: 2) HADGSFSDEMNTILDNLAARDFINWLIQTKITD.

U.S. Pat. Nos. 7,745,403 and 7,563,770 propose the use of GLP-2analogues, including elsiglutide, for preventing or ameliorating sideeffects of chemotherapy, including chemotherapy-induced diarrhea (CID).GLP-2 analogues appear to act in CID by inhibiting enterocyte and cryptcell apoptosis and increasing crypt cell proliferation, thus providingnew cells to replace the damaged intestinal epithelium followingchemotherapy.

A planned experimental trial of elsiglutide was reported onclinicaltrials.gov sometime around Feb. 21, 2012. The official title ofthe trial was Phase II, Double-blind, Randomized, Two-stage,Placebo-controlled Proof of Concept Study in Colorectal Cancer PatientsReceiving 5-FU Based Chemotherapy to Assess the Efficacy of Elsiglutide(ZP1846) Administered s.c. in the Prevention of Chemotherapy InducedDiarrhea (CID). Clinicaltrials.gov reports the following brief summaryof the study: The main objective of this study will be to obtain data onthe efficacy of elsiglutide in preventing Chemotherapy Induced Diarrhea(CID) in patients with colorectal cancer receiving 5-FU basedchemotherapy (FOLFOX4 or FOLFIRI regimen) in comparison to placebo. Theresults of the study are not reported in clinicaltrials.gov, but arereported for the first time herein.

SUMMARY OF THE INVENTION

As specified in the Background Section, there is a great need in the artto prevent/ameliorate toxicities in the gastrointestinal tractassociated with anti-cancer chemotherapies, particularly the CIDresulting from gastrointestinal mucositis. The present inventionaddresses these and other needs by providing the methods andcompositions described below.

The present invention is based on the discovery that the occurrence ofchemotherapy-induced diarrhea (CID) is prevented or its severity isreduced upon administration of the GLP-2 analog elsiglutide. Inparticular the invention is based on the unexpected finding that theadministration of elsiglutide provides a protective effect against CIDthat extends long after the elsiglutide is administered. The effect isespecially pronounced for diarrhea of Grade≧2 as determined by NationalCancer Institute Common Toxicity Criteria for Diarrhea (CTCAE v.4.03).In one human clinical study, 24 mg of elsiglutide was administeredsubcutaneously (s.c.) on days 1, 2, 3 and 4 from the beginning of a 14day chemotherapy cycle. The chemotherapy was administered on days 1 and2 of the cycle. Even though the elsiglutide was administered for only afew days at the beginning of the chemotherapy cycle, the elsiglutide wasable to lower the incidence of Grade 2 and less diarrhea events duringthe entire period of the chemotherapy cycle, including on days 5 through6 when the occurrence of diarrhea is shown to be the greatest, andlevels of citrulline (a marker of intestinal damage) are decreasedcompared to baseline values.

Therefore, in a first embodiment the invention provides a method forpreventing or reducing the occurrence of grade 2 or higher diarrhearesulting from an anti-cancer chemotherapy in a subject in need thereof,which method comprises administering to the subject a therapeuticallyeffective amount of elsiglutide in an elsiglutide regimen, wherein theelsiglutide regimen preferably comprises daily administration ofelsiglutide for four consecutive days, preferably commencing at thestart of the chemotherapy cycle.

More generally speaking, the invention provides a method for treatinggastrointestinal mucositis, or otherwise preventing or reducinggastrointestinal (GI) damage and/or dysfunction associated with ananti-cancer chemotherapy in a subject in need thereof, which methodcomprises administering to the subject a therapeutically effectiveamount of elsiglutide in an elsiglutide regimen, wherein the elsiglutideregimen preferably comprises daily administration of elsiglutide for aplurality of consecutive days, preferably commencing at the start of thechemotherapy cycle and ending prior to the conclusion of thechemotherapy cycle.

Another embodiment relates to the use of elsiglutide to prevent orreducing the occurrence of or severity of gastrointestinal mucositis(including CID) in cancer patients receiving antibody therapy for theircancer, with or without treatment with a small cytotoxic agent. Thisembodiment is particularly useful in preventing or reducing theoccurrence of grade 2 or higher CID. Thus, in still another embodimentthe invention provides a method for preventing or reducinggastrointestinal mucositis in a cancer patient receiving antibodytherapy, including grade 2 or higher CID, by administering to thesubject a therapeutically effective amount of elsiglutide in anelsiglutide regimen, wherein the elsiglutide regimen preferablycomprises daily administration of elsiglutide for four consecutive days,preferably commencing at the start of the antibody cycle.

Still another embodiment relates to the use of elsiglutide to prevent GIdamage as detected by means of certain citrulline levels duringchemotherapy. In this embodiment, the invention provides a method forpreventing GI damage by maintaining citrulline levels in a subjectreceiving chemotherapy, comprising administering to said subject atherapeutically effective amount of elsiglutide.

Additional embodiments and advantages of the invention will be set forthin part in the description which follows, and in part will be obviousfrom the description, or may be learned by practice of the invention.The embodiments and advantages of the invention will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the invention,as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the proportion of patients with diarrhea of any grade, byday on days 1-14, with or without elsiglutide administration in studyTIDE-11-10.

FIG. 2 shows the proportion of patients with diarrhea of grade≧2, by dayon days 1-14, with or without elsiglutide administration in studyTIDE-11-10.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When a peptide active ingredient is referred to herein in its nativeform, it will be understood to include all pharmaceutically acceptablesalts thereof. Thus, references to elsiglutide include elsiglutidehydrochloride, and other pharmaceutically acceptable salts ofelsiglutide.

As used herein, the terms “elsiglutide” and “ZP1846” are usedinterchangeably to refer to a GLP-2 peptide analog having amino acidsequence:

(SEQ ID NO: 1) HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK The terms also encompass peptides provided in the form of a salt. Saltsinclude pharmaceutically acceptable salts such as, e.g., acid additionsalts and basic salts. Non-limiting examples of acid addition saltsinclude hydrochloride salts, citrate salts and acetate salts.Non-limiting examples of basic salts include salts where the cation isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions ⁺N(R³)₃(R⁴), where R³and R⁴ independently designates optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substituted aryl, oroptionally substituted heteroaryl. Other examples of pharmaceuticallyacceptable salts are described in “Remington's Pharmaceutical Sciences”,17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company,Easton, Pa., U.S.A., 1985 and more recent editions, and in theEncyclopaedia of Pharmaceutical Technology.

The terms “cancer chemotherapy” and “chemotherapy” are usedinterchangeably herein to refer to a therapy of cancer by administeringan anti-cancer agent. The terms “anti-cancer agent” and“chemotherapeutic agent” are used herein to refer to any chemicalcompound which is used to treat cancer. Chemotherapeutic agents are wellknown in the art (see, e.g., Gilman A. G., et al., The PharmacologicalBasis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)). Specificnon-limiting examples of chemotherapeutic agents are provided throughoutthe specification and include, for example, FOLFOX (a chemotherapyregimen for treatment of colorectal cancer, which comprisesadministration of folinic acid (leucovorin), fluorouracil (5-FU), andoxaliplatin) and FOLFIRI (a chemotherapy regimen for treatment ofcolorectal cancer, which comprises administration of folinic acid(leucovorin), fluorouracil (5-FU), and irinotecan), as well asadministration of targeted monoclonal antibody therapy (e.g.,bevacizumab, cetuximab, or panitumumab) alone or in combination withchemotherapeutic agents.

The term “chemotherapy cycle” is used herein to refer to a period oftime between the initial administration of an anti-cancer agent and itsrepeat administration. For example, the cycle of the FOLFOX4chemotherapy discussed in the Examples section below includes 14 days,wherein anti-cancer agents are administered only for the first 2 days ofthe cycle as follows: Day 1: oxaliplatin 85 mg/m² IV infusion andleucovorin 200 mg/m² IV infusion both given over 120 minutes at the sametime in separate bags, followed by 5-FU 400 mg/m² IV bolus given over2-4 minutes, followed by 5-FU 600 mg/m² IV infusion as a 22-hourcontinuous infusion; Day 2: leucovorin 200 mg/m² IV infusion, followedby 5-FU 400 mg/m² IV bolus given over 2-4 minutes, followed by 5-FU 600mg/m² IV infusion as a 22-hour continuous infusion. Similarly, the cycleof the FOLFIRI chemotherapy discussed in the Examples section, below,includes 14 days, wherein anti-cancer agents are administered only forthe first 2 days of the cycle as follows: irinotecan (180 mg/m² IV over90 minutes) concurrently with folinic acid (400 mg/m² [or 2×250 mg/m²]IV over 120 minutes), followed by fluorouracil (400-500 mg/m² IV bolus)then fluorouracil (2400-3000 mg/m² intravenous infusion over 46 hours).Bevacizumab is usually given intravenously every 14 days, although thefrequency can be dose dependent (for example 5 mg/kg by intravenousinfusion every two weeks or 7.5 mg/kg by intravenous infusion everythree weeks). In colon cancer, it is given in combination with thechemotherapy drug 5-FU (5-fluorouracil), leucovorin, and oxaliplatin oririnotecan. One recommended dose and schedule for cetuximab is 400 mg/m²administered intravenously as a 120-minute infusion as an initial dose,followed by 250 mg/m² infused over 30 minutes weekly, preferably incombination with FOLFIRI.

The terms “co-administered” and “co-administration” broadly refer toadministration of two or more components, compounds or compositions(e.g., a chemotherapeutic agent and elsiglutide), wherein saidcomponents, compounds or compositions can be administered eithersimultaneously (in one composition or in two or more separatecompositions) or sequentially.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to ±20%,preferably up to ±10%, more preferably up to ±5%, and more preferablystill up to ±1% of a given value. Where particular values are describedin the application and claims, unless otherwise stated, the term “about”is implicit and in this context means within an acceptable error rangefor the particular value.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “prevent,”“prevention,” “treat,” “treatment” and the like are synonymous and meanto reduce the occurrence of a symptom or condition, or to relieve oralleviate at least one symptom associated with such condition, or toslow or reverse the progression of such condition. Within the meaning ofthe present invention, the terms also denotes to arrest, delay the onset(i.e., the period prior to clinical manifestation of a disease) and/orreduce the risk of developing or worsening a disease. For example, inconnection with cancer the term “prevent,” “treat” or “reduce theoccurrence of,” may mean that a therapy has been demonstrated in aprospectively designed clinical trial to reduce the incidence oroccurrence of a clinical endpoint, symptom or condition such as diarrheaor gastrointestinal mucositis.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity uponadministration to a subject in need thereof. Within the context of thepresent invention, when the term “therapeutically effective” is used inconnection with elsiglutide, it refers to an amount of elsiglutide or apharmaceutical composition containing elsiglutide that is effective toprevent side effects or reduce the incidence, occurrence or severity ofside effects of cancer chemotherapy such as damage to thegastrointestinal mucosa or diarrhea. Note that when a combination ofactive ingredients is administered (e.g., a combination of elsiglutideand another compound effective for ameliorating or preventing sideeffects of cancer chemotherapy) the effective amount of the combinationmay or may not include amounts of each ingredient that would have beeneffective if administered individually.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to asubject (e.g., a mammal such as a human). Preferably, as used herein,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inmammals, and more particularly in humans.

As used herein, the term “subject” refers to any mammal. In a preferredembodiment, the subject is human.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

When a range of values can be used to describe a particular regimen, itwill be understood that the range can be defined by selectivelycombining any one of the lower end of variables described in thespecification with any one of the upper end of variables described inthe specification that is mathematically possible.

Throughout this application, whenever a standard is given with referenceto a test or methodology currently accepted and applied in thescientific community, the standard will be understood to be evaluatedwith respect to the test or methodology as it is reported in thepublished literature on Jul. 1, 2014.

Discussion

In a first embodiment the invention provides a method for preventing orreducing the occurrence of grade 2 or higher diarrhea as determined byNational Cancer Institute Common Toxicity Criteria for Diarrhea (CTCAEv.4.03) resulting from an anti-cancer chemotherapy cycle in a subject inneed thereof, which method comprises administering to the subject atherapeutically effective amount of elsiglutide in an elsiglutideregimen, wherein the elsiglutide regimen preferably comprises dailyadministration of elsiglutide, preferably for four consecutive dayscommencing at the start of the chemotherapy cycle.

More generally speaking, the invention provides a method for preventingor reducing gastrointestinal (GI) damage and/or dysfunction associatedwith an anti-cancer chemotherapy cycle in a subject in need thereof,which method comprises administering to the subject a therapeuticallyeffective amount of elsiglutide in an elsiglutide regimen, wherein theelsiglutide regimen preferably comprises daily administration ofelsiglutide for a plurality of consecutive days, preferably commencingat the start of the chemotherapy cycle and ending prior to theconclusion of the chemotherapy cycle.

The invention can also be used to prevent and reduce gastrointestinalmucositis induced by antibody chemotherapy, and in this embodiment theinvention provides a method for preventing gastrointestinal mucositis ina cancer patient receiving antibody therapy alone or in combination withone or more chemotherapy agents, including grade 2 or higher CID, byadministering to the subject a therapeutically effective amount ofelsiglutide in an elsiglutide regimen, wherein the elsiglutide regimenpreferably comprises daily administration of elsiglutide, preferably forfour consecutive days commencing at the start of the antibody cycle.

Still another embodiment relates to the use of elsiglutide to prevent GIdamage as detected by means of certain citrulline levels duringchemotherapy. In this embodiment, the invention provides a method forpreventing GI damage by maintaining citrulline levels in a subjectreceiving chemotherapy, comprising administering to said subject atherapeutically effective amount of elsiglutide. By “maintaining,” it ismeant that the elsiglutide preferably prevents the citrulline levels ofcitrulline from dropping 20% or more, 40% or more, 60% or more, or even80% or more, from the level that citrulline would drop in the absence ofsuch elsiglutide.

Elsiglutide and a chemotherapeutic agent(s)are preferably administeredconcurrently for two or more days, with the elsiglutide administrationbeginning on the same day that the chemotherapy cycle begins, althoughit is feasible to administer or at least initiate the elsiglutideadministration before the administration of the chemotherapeuticagent(s) begins, or to administer elsiglutide after the administrationof the chemotherapeutic agent(s) concludes (i.e., during the days of thechemotherapy cycle when the chemotherapeutic agent(s) is no longeradministered). When the chemotherapy comprises multiple cycles, such as2, 3, 4 or more cycles, elsiglutide is preferably administered duringeach of the cycles. When administered on a daily basis, elsiglutide canbe administered one or more times during the day, but it is preferablyonly administered once daily.

The elsiglutide regimen preferably comprises elsiglutide administrationdaily for 1, 2, 3, 4, 5, or six days of the chemotherapy cycle, oranywhere between these time periods (such as 1-5 days), although 4 daysappears to be adequate. The regimen is also preferably initiated at thestart of the chemotherapy cycle, although the regimen can also beinitiated as many as 1, 2, 3, 4 or 5 days prior to the initiation of thechemotherapy cycle. The regimen is also preferably performed onconsecutive days, although dosing for non-consecutive daily periods canalso be envisioned.

A chemotherapy cycle may comprise administration of chemotherapy for 1or more, 3 or more, 5 or more, 7 or more, 9 or more, or even 10 or moreconsecutive days during the cycle, or anywhere between these timeperiods (such as 1 to up to 5 days). The chemotherapy cycle might lastfor one week, two weeks, three weeks, four weeks, or even more, oranywhere in between these time periods. It has been found herein that alimited period of elsiglutide administration is effective to prevent orreduce the occurrence or severity of gastrointestinal mucositis or CIDthroughout a 14 day chemotherapy cycle, including on days 5 through 9when the incidence of mucositis or CID is shown to be most pronounced.

In a particularly preferred embodiment, the methods are used to preventand reduce the occurrence or severity of more severe cases of CID, grade2 or higher as determined by the National Cancer Institute CommonToxicity Criteria for Diarrhea (CTCAE v.4.03).

According to the present invention, elsiglutide can be used to reducetoxicity of a wide range of different chemotherapeutic agents, prodrugsof such chemotherapeutic agents, and chemotherapy regimens. While somechemotherapy agents and regimens are better known for producing CID anddamaging the gastrointestinal mucosa, the invention can also bepracticed to reduce subclinical occurrences of CID or damage to thegastrointestinal mucosa from practically any chemotherapy agent.Non-limiting examples of such agents include anti-metabolites such aspyrimidine analogs (e.g., 5-fluorouracil [5-FU], floxuridine,capecitabine, gemcitabine and cytarabine) and purine analogs, folateantagonists and related inhibitors (e.g., mercaptopurine, thioguanine,pentostatin and 2-chlorodeoxyadenosine (cladribine));antiproliferative/antimitotic agents including natural products such asvinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine),microtubule disruptors such as taxanes (e.g., paclitaxel, docetaxel),vincristin, vinblastin, nocodazole, epothilones and navelbine,epidipodophyllotoxins (e.g., etoposide, teniposide), DNA damaging agents(e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, nedaplatin,cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,epirubicin, aclarubicin, purarubicin, hexamethyhnelamineoxaliplatin,iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone,nitrosourea, nimustine, ranimustine, estramustine, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics (e.g., dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin), pleomycin,peplomycin, mitomycins (e.g., mitomycin C), actinomycins (e.g.,actinomycin D), zinostatinstimalamer); enzymes (e.g., L-asparaginase);neocarzinostatin; antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (e.g., mechlorethamine,cyclophosphamide and analogs, imidazol carboxamide, melphalan,chlorambucil, nitrogen mustard-N-oxide hydrochloride, ifosfamide),ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa,carboquone, triethylene thiophospharamide), alkyl sulfonates (e.g.,busulfan, isoprosulfan tosylate), nitrosoureas (e.g., carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC); epoxide typecompounds (e.g., mitobronitol); antiproliferative/antimitoticantimetabolites such as folic acid analogs (e.g., methotrexate);platinum coordination complexes (e.g., cisplatin, carboplatin,oxaliplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (e.g., estrogen, tamoxifen, goserelin,bicalutamide, nilutamide) and aromatase inhibitors (e.g., letrozole,anastrozole); anticoagulants (e.g., heparin, synthetic heparin salts andother inhibitors of thrombin); fibrinolytic agents (e.g., tissueplasminogen activator, streptokinase and urokinase), aspirin,dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents;antisecretory agents (e.g., breveldin); immunosuppressives (e.g.,cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine,mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470,genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblastgrowth factor (FGF) inhibitors); angiotensin receptor blockers; nitricoxide donors; antisense oligonucleotides; antibodies (e.g.,trastuzumab); cell cycle inhibitors and differentiation inducers (e.g.,tretinoin); mTOR inhibitors, topoisomerase inhibitors (e.g., doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan,irinotecan); growth factor signal transduction kinase inhibitors(erlotinib, sorafenib, lapatinib); mitochondrial dysfunction inducers;chromatin disruptors; sobuzoxane; tretinoin; pentostatin; flutamide;porphimer natrium; fadrozole; procarbazine; aceglatone, andmitoxantrone.

Non-limiting examples of gastrointestinal (GI) damage and/or dysfunctionassociated with anti-cancer chemotherapies include, for example,chemotherapy-induced diarrhea (CID), nausea, vomiting, anorexia, bodyweight loss, heavy feeling of stomach, constipation, stomatitis, andesophagitis.

The methods of the invention can be used in subjects suffering from abroad range of cancers for which treatment could induce CID orgastrointestinal damage. Non-limiting examples of relevant cancersinclude, e.g., breast cancer, prostate cancer, multiple myeloma,transitional cell carcinoma, lung cancer (e.g., non-small cell lungcancer (NSCLC)), renal cancer, thyroid cancer and other cancers causinghyperparathyroidism, adenocarcinoma, leukemia (e.g., chronic myeloidleukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acutelymphocytic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma,non-Hodgkins lymphoma, Hodgkins lymphoma), head and neck cancer,esophageal cancer, stomach cancer, colon cancer, intestinal cancer,colorectal cancer, rectal cancer, pancreatic cancer, liver cancer,cancer of the bile duct, cancer of the gall bladder, ovarian cancer,uterine endometrial cancer, vaginal cancer, cervical cancer, bladdercancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma,squamous cell cancer, bone cancer, including both primary bone cancers(e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma,malignant fibrous histiocytoma, adamantinoma, giant cell tumor, andchordoma) and secondary (metastatic) bone cancers, soft tissue sarcoma,basal cell carcinoma, angiosarcoma, hemangiosarcoma, myxosarcoma,liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicularcancer, uterine cancer, gastrointestinal cancer, mesothelioma,leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, Waldenstroom'smacroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma,bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,Wilms' tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,retinoblastoma, medullary carcinoma, thymoma, sarcoma, etc.

Methods for Administering Elsiglutide and Elsiglutide Compositions

Specific elsiglutide doses useful in the methods of the invention willdepend on the type of chemotherapy side effects to be treated, theseverity and course of these side effects, previous therapy, thepatient's clinical history and response to chemotherapy and elsiglutide,as well as the discretion of the attending physician. In one specificembodiment, such doses range from 5 to 80 or from 10 to 40 mg/ day.

The administration of elsiglutide according to the methods of theinvention can be performed by any suitable route. Specific non-limitingexamples of useful routes of administration include subcutaneous,intravenous (IV), intraperitoneal (IP), and intramuscular.

In certain embodiments, elsiglutide is formulated in a pharmaceuticalcomposition with a pharmaceutically acceptable carrier or excipient. Incertain embodiments, elsiglutide is combined in a pharmaceuticalcomposition together with another compound effective for ameliorating orpreventing side effects of cancer chemotherapy. The formulations used inthe methods of the invention may conveniently be presented in unitdosage form and may be prepared by methods known in the art. The amountof active ingredients that can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated and the particular mode of administration. The amount of activeingredients that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect.

In general, the formulations can be prepared with a liquid carrier, or afinely divided solid carrier, or both, and then, if necessary, shapingthe product. Pharmaceutical compositions suitable for parenteraladministration may comprise elsiglutide in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

Example 1 Initial Clinical Characterization of Elsiglutide

1.1. Summary of Clinical Data

Data on human exposure to elsiglutide generated so far originate fromthree clinical trials, one performed in healthy subjects (study 06-013)and two in cancer patients (TIDE-09-04, TIDE-11-10). The main objectiveof the first two studies was the evaluation of the safety, tolerabilityand maximum tolerated dose (MTD) of ascending elsiglutide dosesadministered as s.c. (both studies) and i.v. (only study 06-013) bolusinjections. In study 06-013 elsiglutide was administered as single i.v.or s.c. bolus, while in study TIDE-09-04 each dose was administered ass.c. bolus on 4 consecutive days. Study TIDE-11-10 was a phase II proofof concept study that mainly evaluated the efficacy of elsiglutideadministered as 24 mg daily s.c. bolus injections for 4 consecutive daysin preventing CID in patients with colorectal cancer receiving 5-FUbased chemotherapy (FOLFOX4 or FOLFIRI regimen). The secondaryobjectives of all three studies included the evaluation of thepharmacokinetics of elsiglutide and its major metabolites in humans.

Pharmacokinetics

Elsiglutide has a short half-life (0.4 h) and is quickly eliminated fromblood circulation after single i.v. administration. Data, althoughlimited to 1 subject only, indicate an absolute bioavailability of0.29%. The two human metabolites of elsiglutide, ZP2242 and ZP2712, havelonger half-lives (6-9 h) with overall plasma levels substantiallyhigher than elsiglutide. ZP2242 is the major metabolite and revealed anAUC at least 10 times higher than that of ZP2712. In general, exposureto the metabolite ZP2242 was >25-fold higher than exposure to the parentZP1846 (elsiglutide) on Day 1. Exposure to the metabolite ZP2712 was >4fold higher than exposure to the parent ZP1846 (elsiglutide) on Day 1.

In study TIDE-09-04 the pharmacokinetics of elsiglutide and themetabolites ZP2242 and ZP2712 appeared to be dose independent, i.e.,Cmax and AUC increases were proportional to the dose increase althoughCmax for elsiglutide increased less than expected on Day 4 at dosesabove 60 mg/day. The t1/2Z, CL/F and Vz/F were dose independent.

Study TIDE-11-10 showed that the pharmacokinetics of elsiglutide and itsmetabolites ZP2242 and ZP2712 varied considerably across patients andover multiple days.

Overall, PK data generated in the framework of Phase 1 and Phase 2astudies, indicated a rapid elimination of both the parent compound andits metabolites, which allow to predict a very modest, if any,accumulation at steady state.

Safety

Safety data in humans are derived from two phase I dose-escalationstudies and one phase II study, which included 202 human subjects (36healthy subjects and 166 colon and colorectal cancer patients receiving5-FU-based chemotherapy), 117 of whom received active treatment.

In the first dose-escalation study (06-013) in healthy volunteers, themaximum tolerated dose (MTD; defined in this study as the dose levelimmediately prior to the dose level at which further dose escalation washalted due to the study ‘Stopping Rules’) was 9.6 mg i.v. and 3 mg s.c.,as an AE that met the study stopping rules was observed in the 19.2 mgi.v. group (moderate positional lightheadedness) and in the 6 mg s.c.group (moderate systolic hypotension). Thirteen of 27 subjects includedin this study experienced at least 1 treatment-emergent adverse event(TEAE) judged by the investigator as having either a possible, probableor definitive relation to the study medication. The most commonlyreported related TEAEs being postural dizziness, nausea, and injectionsite erythema.

In the second dose-escalation study (TIDE-09-04) in cancer patients anindependent data safety monitoring board supervised the dose escalationprocess. A dose of 93 mg/day administered for 4 consecutive days wasreached and no dose limiting toxicities occurred, proving thatelsiglutide was better tolerated than foreseen after study 06-013.

In studies TIDE-09-04 and TIDE-11-10 the overall pattern of TEAEs was asexpected for patients with cancer receiving chemotherapy. In bothstudies the safety profiles of elsiglutide and placebo were generallysimilar. More patients in the elsiglutide group reported TEAEs comparedto patients in the placebo group, mostly due to injection site reactionsin the elsiglutide group. No serious or severe injection site reactionswere observed.

In study TIDE-09-04, out of the 28 treated patients (21 on activetreatment and 7 on placebo), 9 (32.1%; 7 patients in elsiglutide groupand 2 patients in placebo group) experienced at least one TEAE judged bythe investigator to be at least possibly related to treatment. In studyTIDE-11-10, out of the 138 treated patients (69 on elsiglutide and 69 onplacebo), 8 (11.6%), all in the elsiglutide group, had at least one TEAEjudged by the investigator to be at least possibly related tostudy-drug. The most frequent related TEAEs overall in both studies wereinjection site events (in particular injection site erythema andinjection site pain) followed by constipation.

In all three studies clinical laboratory, vital sign, ECG, and physicalexamination data did not reveal any clinically significant abnormalfinding. No deaths or SAEs related to the study drug were reported.

Hence, on the basis of the human safety data collected so far, no safetyconcerns have been raised.

TIDE-11-10 Phase II Proof of Concept Study to Evaluate the Efficacy ofElsiglutide Administered as 24 mg Daily s.c. Bolus Injections for 4Consecutive Days in Preventing CID in Patients with Colorectal CancerReceiving 5-FU Based Chemotherapy (FOLFOX4 or FOLFIRI Regimen)

The formulation used in clinical trials is a lyophilized sterile powderfor s.c. administration after reconstitution with sterile water forinjection. The efficacy results obtained in study TIDE-11-10 indicatethat elsiglutide had a preventive effect on the occurrence of grade ≧2diarrhea (see FIGS. 1 and 2). This study also included an evaluation ofthe levels of citrulline, an amino acid mainly produced by enterocytes,a decrease of which is indicative of an intestinal mucosal damagefollowing chemotherapy. Objectives: The main objective of TIDE-11-10proof of concept study was to obtain data on the efficacy of elsiglutidein preventing CID in patients with colorectal cancer receiving 5-FUbased chemotherapy (FOLFOX4 or FOLFIRI regimen) in comparison toplacebo. In addition, safety and tolerability of the administeredrepeated doses of elsiglutide were evaluated, and the pharmacokinetics(PK) of elsiglutide and its metabolites ZP2242 and ZP2712 wereinvestigated in a subset of patients in each treatment arm.

Methodology: This was a phase II, multicenter, double-blind, randomized,placebo-controlled, two-stage, proof of concept study with an interimfutility analysis in colorectal cancer patients receiving 5-FU-basedchemotherapy (FOLFOX4 or FOLFIRI) and administered elsiglutidesubcutaneously (s.c.) for 4 consecutive days.

138 patients received a daily dose of 24 mg elsiglutide (or placebopractically identical in composition to the active study drug) via asingle s.c. injection for 4 consecutive days, starting from the firstday of chemotherapy administration. The patients were hospitalized atleast until Day 3. Further visits were scheduled for Days 4, 5 and 15and for a Follow-up Visit on Day 28-32. Safety and tolerability weremonitored throughout the study.

Diagnosis and Main Criteria for Inclusion: Female and male patients ofat least 18 years of age with confirmed diagnosis of colorectal cancerand an Eastern Cooperative Oncology

Group (ECOG) performance status ≦2, chemotherapy-naïve, and scheduled toreceive a FOLFOX4 or FOLFIRI chemotherapy regimen.

ECOG PERFORMANCE STATUS* Grade ECOG 0 Fully active, able to carry on allpre-disease performance without restriction 1 Restricted in physicallystrenuous activity but ambulatory and able to carry out work of a lightor sedentary nature, e.g., light house work, office work 2 Ambulatoryand capable of all selfcare but unable to carry out any work activities.Up and about more than 50% of waking hours 3 Capable of only limitedselfcare, confined to bed or chair more than 50% of waking hours 4Completely disabled. Cannot carry on any selfcare. Totally confined tobed or chair 5 Dead *As published in Oken et al., Toxicity And ResponseCriteria Of The Eastern Cooperative Oncology Group, Am J Clin Oncol 5:649-655, 1982.

Efficacy: The endpoint of primary interest was:

-   -   Number of patients experiencing no diarrhea from Day 1 to Day 14

Secondary endpoints were:

-   -   Proportion of patients experiencing grades ≧2 diarrhea at each        day from Day 1 to Day 14 according to National Cancer Institute        Common Terminology Criteria for Adverse Events (NCI-CTCAE v.        4.03);    -   Worst grade of diarrhea according to NCI-CTCAE at each day from        Day 1 to Day 14;    -   Time to occurrence of diarrhea, defined as the first day in        which a grade ≧1 diarrhea was assessed (from Day 1 to day 14);    -   Number of days with presence of grade ≧1 diarrhea (from Day 1 to        Day 14);    -   Number of days with presence of grade ≧2 diarrhea (from Day 1 to        Day 14);    -   Number of days with presence of at least one bowel movement        accompanied by urgency (from Day 1 to 14);    -   Number of days with presence of at least one episode of fecal        incontinence (from Day 1 to 14);    -   Proportion of patients who required i.v. fluids due to CID (from        Day 1 to 14);    -   Proportion of patients who required changes to the primary        therapy (chemotherapy dose reduction, delay or change to        regimen) due to CID as of Day 2, Day 14 and as of Day 28;    -   Proportion of patients who used rescue medication (i.e.        medication used for treatment of diarrhea) from Day 1 to Day 14.

In addition, the proportion of patients who were limited concerningself-care Activities of Daily Living (ADL), the number of stools perday, the number of bowel movements accompanied by urgency per day, andthe number of episodes of fecal incontinence per day were summarized forDay 1 to Day 14. Mean blood concentrations of citrulline (a biomarkerfor intestinal integrity, a decrease being indicative of an intestinalmucosal damage following chemotherapy) were summarized by treatmentgroup for baseline, Day 5, and Day 15 including changes compared tobaseline.

Summary—Conclusions:

For the overall trial, i.e. Stage 1 and Stage 2, (69 patients in eachtreatment group), superiority of elsiglutide to placebo was to beconcluded if the difference in the number of responders(elsiglutide—placebo) was larger than or equal to 5. More patients wereresponders, i.e. had no diarrhea, in the elsiglutide group (43 patients)than in the placebo group (39 patients). While a trend towardsuperiority was shown, superiority of elsiglutide over placebo could notbe statistically demonstrated.

Number of Responders¹ in the Period from Day 1 to Day 14 Intent-to-treatSet Placebo 24 mg/day Elsiglutide N = 69 N = 69 n (%) n (%) Responder 39(56.5) 43 (62.3) Non-responder 30 (43.5) 26 (37.7) ¹Responder wasdefined as a patient experiencing no diarrhea N = number of patients intreatment group, n = number of patients with data available, % =percentage based on N.

A higher frequency of diarrhea grade ≧2 was observed in the placebogroup (15 patients) compared to the elsiglutide group (5 patients): thedifference was more evident at Days 5, 6, and 7. See FIG. 2.

Grade of Diarrhea According to NCI-CTCAE version 4.03 (Day 1 to Day 14)Full Analysis Set Placebo 24 mg/day Elsiglutide N = 69 N = 69 Grade¹ n(%) n (%) Grade 0 39 (56.5) 43 (62.3) Grade 1 15 (21.7) 21 (30.4) Grade2 14 (20.3) 4  (5.8) Grade 3 1  (1.4) 1  (1.4) Grade <2 54 (78.3) 64(92.8) Grade ≧2 15 (21.7) 5  (7.2) ¹Worst grade of diarrhea Day 1 to Day14 NCI-CTCAE = National Cancer Institute Common Terminology Criteria forAdverse Events, % = percentage based on N.

In both treatment groups, the incidence of diarrhea increased on Day 2and remained high up to Day 9. Diarrhea occurred most often 5 to 8 daysafter the first administration of chemotherapy.

Mean citrulline levels were similar in the treatment groups at baseline(placebo, 32.7 μmol/L; elsiglutide, 33.5 μmol/l). Mean levels decreasedbetween Baseline and Day 5 in both treatment groups, with a lesspronounced decrease in the elsiglutide group than in the placebo group.Between Day 5 and Day 15, mean citrulline levels increased in bothtreatment groups. In the elsiglutide group, mean citrulline levels atDay 15 were slightly increased compared to baseline (Day 15: placebo,29.7 μmol/l; elsiglutide, 36.5 μmol/l).

Example 2 Follow-On Clinical Trial to Determine Optimal Dose and Regimenfor Elsiglutide Effect on CID

TIDE-13-22: Randomized, Double Blind, Parallel Group,Placebo-Controlled, Dose Finding Study in Colorectal Cancer PatientsReceiving 5-FU Based Chemotherapy to Assess the Efficacy of DifferentDoses of s.c. Elsiglutide in the Prevention of Chemotherapy InducedDiarrhea (CID)

The use of chemotherapy regimens for colorectal cancer treatment evolvescontinuously in all countries. In order to have a homogeneous patientpopulation, the present study is planned to be conducted on patientswith colorectal cancer scheduled to receive any FOLFOX (fluorouracil,folinic acid and oxaliplatin) or FOLFIRI (fluorouracil, folinic acid andirinotecan) chemotherapy regimens. These regimens have been described tocause CID in 30-80% of treated patients (Cherny, J Pain Symptom Manage2008; 36:413-23; Arnold et al., J Support Oncol 2005; 3:227-232;Tournigand et al., J Clin Oncol. 2004; 22:229-237). Considering thatmonoclonal antibodies are often used together with standard chemotherapyregimens, this study also includes an additional patient population thatis scheduled to receive FOLFOX or FOLFIRI together with monoclonalantibodies such as bevacizumab, cetuximab, panitumumab, or others, inorder to gather preliminary results in this patient population. Thepresent study aims at further investigating the efficacy of elsiglutideand identifying the most appropriate s.c. dosage. The dosages for thepresent study are 10 mg/day, 20 mg/day, and 40 mg/day each administereds.c. on 4 consecutive days.

Pharmacological preclinical studies in rats have shown that at a dose of400 nmol/kg elsiglutide prevents 5-Fluorouracil (5-FU)-induced smallintestinal atrophy and diarrhea, attenuates body weight loss anddecreases mortality in rats. Elsiglutide also decreases the severity ofirinotecan-induced diarrhea, including late-onset diarrhea, decreasesbody weight loss and lethality and enhances animal recovery. Moreover,elsiglutide has an intestinotrophic effect and histopathologicalobservations show that it drastically reduces irinotecan induced severeintestinal damage. The dose of 400 nmol/kg corresponds to 1.7 mg/kgwhich in term of HED (human equivalent dose) results in approximately 20mg (see guidance “Estimating the Maximum Safe Starting Dose in InitialClinical Trials for Therapeutics in Adult Healthy Volunteers”, FDA July2005). In addition PK/pharmacodynamic (PD) data in different animalspecies indicate that a dose of 20 mg/day could be considered the lowestefficacious dose.

The phase I and II studies described above (TIDE-09-04 and TIDE-11-10)have shown that the tolerability of the product is better thanoriginally expected based on study 06-013. In study TIDE-09-04 a dose of93 mg/day on 4 consecutive days was reached with no dose limitingtoxicity. On the basis of pharmacological and clinical data (TIDE-09-04and TIDE-11-10), it was deemed appropriate in the present study toconsider a 4-day administration, starting from the day of chemotherapy.In study TIDE-11-10 a 24 mg/day dose administered s.c. on 4 consecutivedays has shown some evidence of efficacy.

The present study aims at further investigating the efficacy ofelsiglutide and identifying the most appropriate s.c. dosage. Thedosages are 10 mg/day, 20 mg/day, and 40 mg/day each administered s.c.on 4 consecutive days.

A placebo treatment is included in this study as a control group inwhich the incidence of CID can be assessed. Moreover, besides clinicalevaluation of the occurrence of diarrhea, the plasma level ofcitrulline, is also evaluated in order to establish its potential roleas a biomarker of mucosal repair. Reduced citrulline levels aresuggestive of intestinal failure (Crenn et al., Clin Nutr. 2008;27(3):328-339) and have also been observed to be related with intestinalmucosal damage following chemotherapy (Herbers et al., Ann Oncol. 2010;21(8):1706-1711).

Study Objectives

The primary objective of the present study is to compare the efficacy of3 s.c. doses of elsiglutide vs placebo and vs each other in theprevention of CID in colorectal cancer patients treated with 5-FU basedchemotherapy (FOLFOX or FOLFIRI) with no addition of a monoclonalantibody.

As a secondary objective, the efficacy of 3 s.c. doses of elsiglutide vsplacebo and vs each other in the prevention of CID in colorectal cancerpatients treated with 5-FU based chemotherapy (FOLFOX or FOLFIRI) givenin combination with a monoclonal antibody is explored.

Investigational Plan

This is a randomized, stratified, double-blind, double-dummy, parallelgroup, placebo-controlled, dose finding, multicenter, multinational,phase II study in patients with colorectal cancer receiving 5-FU-basedchemotherapy (FOLFOX or FOLFIRI) to assess the efficacy of differentdoses of s.c. elsiglutide in the prevention of CID. Chemotherapy naïveor non-naïve patients receive, starting from the day of chemotherapyadministration, a single daily dose subcutaneously (s.c.) of elsiglutide10, 20 or 40 mg or placebo for 4 consecutive days. Each patient is inthe study for 3 consecutive chemotherapy cycles (14 days each). Thetreatment period for each patient is 4 consecutive days at each of thefirst two chemotherapy cycles.

The study includes 480 patients receiving 5-FU-based chemotherapy(FOLFOX or FOLFIRI) (“Target population”), and an additional group of upto 120 patients receiving the 5 FU-based chemotherapy in combinationwith a monoclonal antibody (“Additional population”).

TABLE 1 Study populations and treatment groups 5-FU-based 5-FU-basedchemotherapy chemotherapy + MAb Treatment group: Target populationAdditional population Placebo 120 patients Max 30 patients 10 mg/dayelsiglutide 120 patients Max 30 patients 20 mg/day elsiglutide 120patients Max 30 patients 40 mg/day elsiglutide 120 patients Max 30patients Total per population 480 patients Max 120 patients  Totaloverall Max 600 patients

Within each population, patients are randomly allocated to receive oneof 4 treatments (3 doses of elsiglutide or placebo) at Day 1, before thestart of chemotherapy. The randomization is stratified by chemotherapyregimen (FOLFOX or FOLFIRI) and by country.

Inclusion Criteria

-   -   1. Written informed consent;    -   2. Male or female patient >18 years of age;    -   3. Histologically or cytologically confirmed diagnosis of        colorectal cancer;    -   4. Patients scheduled to receive at least 3 consecutive cycles        of the same regimen of FOLFOX or FOLFIRI        (Oxaliplatin/Irinotecan, Folinic acid, 5-FU).    -   5. Only for the Additional Population: Patient scheduled to        receive monoclonal antibodies in combination with FOLFOX or        FOLFIRI chemotherapy regimen;    -   6. A performance status of ≦2 according to the Eastern        Cooperative Oncology Group (ECOG) scale;    -   7. Non-childbearing female patient or female patient of        childbearing potential using reliable contraceptive measures and        having negative pregnancy test before treatment administration;    -   8. Able to read, understand, follow the study procedure and        complete patient diary.

Efficacy Assessments

Patients preferably exhibit efficacy on the basis of one or more of theprimary or secondary efficacy endpoints, as described below. Theefficacy assessment is based both on data recorded by the patient and onthe clinical assessment of the Investigator. Patients are asked torecord on a daily basis in a e-Diary information related to her/hisbowel movements (including time, number and consistency of stools(following the Bristol Stool Form Scale as described in Lewis andHeaton, Scand J Gastroenterol. 1997; 32 (9):920-924), urgency and fecalincontinence), limitations in activity of daily living (ADL), use ofrescue medications due to diarrhea and abdominal discomfort. Moreover,patients are requested to report daily the occurrence of diarrhea. Thepatient e-Diary is to be filled in daily from Day 1 to Day 14 of Cycles1, 2 and 3.

The occurrence of events of diarrhea over the 14-day period followingeach chemotherapy is assessed by the investigator based on theinformation collected in the patient's eDiary; severity of each event ofdiarrhea is graded by the Investigator according to NCI-CTCAE v.4.03scale. The maximum grade assigned to any of the individual events isidentified. A maximum Grade ≧2 diarrhea is considered for the primaryendpoint.

In addition, the Investigator assigns a unique grade to the whole 14-dayperiod (“overall grade”, based on NCI-CTCAE v.4.03 scale).

Plasma levels of citrulline are measured in all patients to evaluate itspotential mucosa-protective effect. For this purpose, blood samples aretaken at Screening, Day 2 of Cycle 1 (before study drug administration),Day 5 and Day 15 of the first 2 chemotherapy cycles, i.e. when the studydrug is administered, and at Follow-up.

Safety is assessed based on adverse events (AEs), physical examinations,vital signs, clinical laboratory test results, immunogenicity data and12-lead electrocardiograms (ECGs).

The PK of elsiglutide and its active metabolites ZP2242 and ZP2712 isassessed in all patients randomized to each of the study treatments andconsenting to participate in PK sampling. The individual's dense plasmaconcentration-time data is evaluated and the standard PK parameters areestimated. The dose-proportionality is also investigated in the testeddose range between 10 and 40 mg. The influence of possible demographicand therapeutic covariates on the PK parameters and their variability isinvestigated by both a two-stage population PK approach and non-linearmixed effect modeling. The possible relationship between exposure toelsiglutide and its metabolites and efficacy measures is explored.

Patient's health related quality of life is measured by using theEQ-5D-3L questionnaire.

(a) Primary Efficacy Endpoint

Proportion of patients experiencing a maximum Grade ≧2 diarrhea duringthe first cycle of chemotherapy (from Day 1 to Day 14 of Cycle 1) inTarget population.

(b) Secondary Efficacy Endpoints

Proportion of patients experiencing a maximum Grade ≧2 diarrhea duringthe first cycle of chemotherapy in the Additional population.

The following endpoints are considered for the Target population. Theyare also considered for the Additional population as relevant based onthe number of patients available.

-   -   Proportion of patients experiencing a maximum Grade ≧2 diarrhea        during the second and third cycle of chemotherapy;    -   Proportion of patients experiencing a maximum Grade ≧2 diarrhea        over the first two cycles of chemotherapy (i.e. in at least one        of the first two cycles);    -   Proportion of patients experiencing a maximum Grade 1, Grade 2,        Grade 3, Grade 4, Grade 5 and any Grade (i.e. ≧1) diarrhea by        cycle (Cycle 1, 2 and 3);    -   Proportion of patients experiencing an overall Grade ≧2 diarrhea        by cycle (Cycle 1, Cycle 2 and Cycle 3);    -   Proportion of patients experiencing an overall Grade ≧2 diarrhea        over the first two cycles of chemotherapy (i.e. in at least one        of the first two cycles);    -   Proportion of patients experiencing an overall Grade 1, Grade 2,        Grade 3, Grade 4, Grade 5 and any Grade (i.e. ≧1) by cycle        (Cycle 1, 2 and 3);    -   Time to onset of first event of diarrhea of any Grade (i.e. ≧1)        and time to onset of first event of diarrhea of Grade ≧2 (as        assessed by the Investigator) by cycle (Cycle 1, 2 and 3);    -   Time to first day with diarrhea (as reported by patient in the        eDiary) by cycle (Cycle 1, 2 and 3);    -   Cumulative duration (days) of any Grade (i.e. ≧1) diarrhea        events and cumulative duration of Grade ≧2 diarrhea events (as        assessed by the Investigator) by cycle (Cycle 1,2 and 3);    -   Cumulative duration (days) of diarrhea events (as assessed by        the Investigator) by grade (Grade 1, Grade 2, Grade 3, Grade 4,        Grade 5) and by cycle (Cycle 1, 2 and 3);    -   Number of events of diarrhea by grade (as assessed by the        Investigator) by cycle (Cycle 1,2 and 3);    -   Number of days with presence of diarrhea (as reported by patient        in the eDiary) by cycle (Cycle 1, 2 and 3);    -   Number of days with presence of at least one bowel movement with        stools of consistency 6 or 7 (according to Bristol Stool Form        Scale) accompanied by urgency or by fecal incontinence by cycle        (Cycle 1, 2 and 3);    -   Number of days with presence of abdominal discomfort by cycle        (Cycle 1, 2 and 3);    -   Number of days with limitation of self-care activities due to        diarrhea by cycle (Cycle 1, 2 and 3);    -   Proportion of patients: who required i.v. fluids due to CID, who        required changes to the primary therapy (chemotherapy dose        reduction, delay or change to regimen) due to CID, who used        rescue medication (i.e. medication used for treatment of        diarrhea) by cycle (Cycle 1, 2 and 3);    -   Proportion of patients having a maximum Grade ≧2 diarrhea—shift        from Cycle 1 to Cycle 2 and from Cycle 2 to Cycle 3;    -   Proportion of patients having a maximum Grade ≧1 diarrhea—shift        from Cycle 1 to Cycle 2 and from Cycle 2 to Cycle 3;    -   Proportion of patients having an overall Grade ≧2 diarrhea—shift        from Cycle 1 to Cycle 2 and from Cycle 2 to Cycle 3;    -   Proportion of patients having an overall Grade ≧1 diarrhea—shift        from Cycle 1 to Cycle 2 and from Cycle 2 to Cycle 3;    -   Time trend of the citrulline plasma concentrations in Cycles 1,        2 and 3.

(c) Evaluation of the Severity of Diarrhea

Severity of diarrhea is classified by the Investigator according to theNCI-CTCAE, Version 4.03 (June 2010) as described in 2 below:

TABLE 2 National Cancer Institute Common Toxicity Criteria for Diarrhea(CTCAE v. 4.03) Adverse event: Diarrhea Grade 1 Grade 2 Grade 3 Grade 4Grade 5 Increase Increase Increase of ≧7 Life- Death of <4 stools/ of4-6 stools/day over threatening day over stools/ baseline; consequences;baseline; day over incontinence; urgent mild increase baseline;hospitalization intervention in ostomy* moderate indicated; indicatedoutput increase in severe increase compared to ostomy* in ostomy*baseline output output compared compared to to baseline; baselinelimiting self care ADL# Definition: a disorder characterized by frequentand watery bowel movements. A Semi-colon indicates ‘or’ within thedescription of a grade *Patients with any type of ostomy were excludedfrom the present study #Self care ADL refers to bathing, dressing andundressing, feeding self, using the toilet, taking medication, and notbedridden.

(d) Quality of Life Assessment

Patient's health related quality of life is measured using the EQ-5D-3Lquestionnaire developed by the EuroQol Group. Patients complete thequestionnaire EQ-5D-3L at Screening, Day 5 and Day 15 of Cycles 1, 2 and3 (Follow-Up). The questionnaire is part of the e-Diary. The EQ-5D 3Lhas been designed as an international, standardized, generic instrumentthat describes health status in 5 dimensions. It generates 3 types ofdata for each patient:

-   -   1. a profile indicating the extent of problems across the 5        dimensions    -   2. a weighted health index based on general population values    -   3. a score on the self-rated “thermometer”, indicating the        patient's own assessment of their health status

The main objective of this evaluation is to assess whether or notelsiglutide at different dosages vs. placebo is associated with apositive impact in patients' HRQL from baseline.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

1. A method for preventing or reducing the occurrence or severity ofgrade 2 or higher diarrhea resulting from an anti-cancer chemotherapy ina subject in need thereof, which method comprises administering to thesubject a therapeutically effective amount of elsiglutide in anelsiglutide regimen, wherein the elsiglutide regimen preferablycomprises daily administration of elsiglutide for four consecutive dayscommencing at the start of the chemotherapy cycle.
 2. A method ofpreventing or reducing gastrointestinal (GI) damage and/or dysfunctionresulting from an anti-cancer chemotherapy in a subject in need thereof,which method comprises administering to the subject a therapeuticallyeffective amount of elsiglutide in an elsiglutide regimen, wherein theelsiglutide regimen comprises daily administration of elsiglutide for aplurality of consecutive days preferably commencing at the start of thechemotherapy cycle and ending prior to the conclusion of thechemotherapy cycle.
 3. The method of claim 1, wherein said chemotherapycomprises antibody therapy with or without small-molecule chemotherapy.4. The method of claim 1, wherein said chemotherapy comprisesbevacizumab, cetuximab or panitumumab antibody therapy with or withoutsmall-molecule chemotherapy.
 5. The method of claim 1, wherein saidelsiglutide regimen comprises daily administration of elsiglutide for 2to 6 days, and said chemotherapy cycle is 8 to 24 days.
 6. The method ofclaim 1, wherein said elsiglutide regimen comprises daily administrationof elsiglutide for 4 days.
 7. The method of claim 1, wherein theelsiglutide regimen prevents or reduces the occurrence of grade 2 orhigher diarrhea resulting from an anti-cancer chemotherapy on days 5and/or 6 of a chemotherapy cycle.
 8. The method of claim 2, wherein theelsiglutide regimen reduces GI damage and/or dysfunction on days 5and/or 6 of a chemotherapy cycle.
 9. The method of claim 2, wherein theGI damage and/or dysfunction associated with the anti-cancerchemotherapy is gastrointestinal mucositis or chemotherapy-induceddiarrhea (CID).
 10. The method of claim 9, wherein the CID is Grade ≧2diarrhea as determined by National Cancer Institute Common ToxicityCriteria for Diarrhea (CTCAE v.4.03).
 11. The method of claim 1, whereinthe elsiglutide is administered during at least the first fourconsecutive days from the beginning of the chemotherapy cycle.
 12. Themethod of claim 1, wherein the elsiglutide is administered for twocycles of chemotherapy during the first four consecutive days from thebeginning of each chemotherapy cycle.
 13. The method of claim 1, whereinthe chemotherapy cycle is up to 14 days long.
 14. The method of claim 1,wherein the chemotherapy cycle is 14 days or longer.
 15. The method ofclaim 1, wherein the therapeutically effective amount of the elsiglutideis about 10-40 mg/day.
 16. The method of claim 1, wherein thetherapeutically effective amount of elsiglutide is selected from about10 mg/day, about 20 mg /day, and about 40 mg/day.
 17. The method ofclaim 1, wherein the anti-cancer chemotherapy comprises administrationof one or more compounds selected from the group consisting ofantimetabolites, alkylating agents, anticancer antibiotics,microtubule-targeting agents, topoisomerase inhibitors, alkaloids,antibodies, pyrimidine analogs, purine analogs, folate antagonists,epidipodophyllotoxins, DNA damaging agents, antiplatelet agents,platinum coordination complexes, hormones, hormone analogs, aromataseinhibitors, anti-angiogenic compounds, growth factor inhibitors,angiotensin receptor blockers, nitric oxide donors, antisenseoligonucleotides, cell cycle inhibitors, differentiation inducers, mTORinhibitors, mitochondrial dysfunction inducers, chromatin disruptors.18. The method of claim 1, wherein the anti-cancer chemotherapycomprises administration of one or more compounds selected from thegroup consisting of 5-fluorouracil (5-FU), floxuridine, capecitabine,gemcitabine, cytarabine, irinotecan, doxorubicin (adriamycin),amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide,epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, lapatinib,oxaliplatin, cisplatin, carboplatin, folinic acid, methothrexate,erlotinib, sorafenib, and lapatinib.
 19. The method of claim 1, whereinthe anti-cancer chemotherapy comprises administration of oxaliplatin oririnotecan.
 20. The method of claim 1, wherein the anti-cancerchemotherapy comprises administration of oxaliplatin or irinotecan incombination with cetuximab, bevacizumab, and/or panitumumab.
 21. Themethod of claim 1, wherein anti-cancer chemotherapeutic agent(s) isadministered at least during the first two consecutive days from thebeginning of each chemotherapy cycle.
 22. The method of claim 1, whereinthe anti-cancer chemotherapy is administered as FOLFOX or FOLFIRIchemotherapy regimen.
 23. The method of claim 1, wherein the elsiglutideis administered subcutaneously (s.c.).
 24. The method of claim 1,wherein the elsiglutide is administered intravenously orintraperitoneally.
 25. The method of claim 1, wherein the subject is ahuman.
 26. The method of claim 1, wherein the subject has a cancer withperformance status of ≦2 according to the Eastern Cooperative OncologyGroup (ECOG).
 27. The method of claim 1, wherein the subject ischemotherapy-naïve prior to the start of the first chemotherapy cycle.28. The method of claim 1, further comprising measuring blood levels ofcitrulline in said subject before and after the elsiglutideadministration.
 29. A method of preventing or reducing gastrointestinal(GI) damage and/or dysfunction resulting from an administration of ananti-cancer chemotherapeutic agent in a subject in need thereof, whichmethod comprises administering to the subject a therapeuticallyeffective amount of elsiglutide in an elsiglutide regimen, wherein theelsiglutide regimen comprises daily administration of elsiglutide for aplurality of consecutive days commencing before, during or after theadministration of the anti-cancer chemotherapeutic agent.
 30. The methodof claim 29, wherein the administration of elsiglutide is commencedprior to the administration of the anti-cancer chemotherapeutic agent.31. The method of claim 29, wherein the administration of elsiglutide iscommenced during the administration of the anti-cancer chemotherapeuticagent.
 32. The method of claim 29, wherein the administration ofelsiglutide is commenced after the administration of the anti-cancerchemotherapeutic agent is completed.
 33. A method for preventing orreducing GI damage by maintaining citrulline levels in a subjectreceiving chemotherapy, comprising administering to said subject atherapeutically effective amount of elsiglutide.
 34. The method of claim33, wherein said chemotherapy comprises a cycle of chemotherapy, andsaid therapeutically effective amount of elsiglutide comprises dailyadministration of elsiglutide for a plurality of consecutive daysbeginning at the initiation of the cycle of chemotherapy.